Showing papers on "Biofilm matrix published in 2019"

Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations.

Abstract: The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.

Nanoparticles for Oral Biofilm Treatments.

Abstract: Pathogenic oral biofilms are universal, chronic, and costly. Despite advances in understanding the mechanisms of biofilm formation and persistence, novel and effective treatment options remain scarce. Nanoparticle-mediated eradication of the biofilm matrix and resident bacteria holds great potential. In particular, nanoparticles that target specific microbial and biofilm features utilizing nontoxic materials are well-suited for clinical translation. However, much work remains to characterize the local and systemic effects of therapeutic agents that are topically applied to chronic biofilms, such as those that cause dental caries. In this Perspective, we summarize the pathogenesis of oral biofilms, describe current and future nanoparticle-mediated treatment approaches, and highlight outstanding questions that are paramount to answer for effectively targeting and treating oral biofilms.

The Pseudomonas aeruginosa lectin LecB binds to the exopolysaccharide Psl and stabilizes the biofilm matrix.

Abstract: Pseudomonas aeruginosa biofilms are composed of exopolysaccharides (EPS), exogenous DNA, and proteins that hold these communities together. P. aeruginosa produces lectins LecA and LecB, which possess affinities towards sugars found in matrix EPS and mediate adherence of P. aeruginosa to target host cells. Here, we demonstrate that LecB binds to Psl, a key matrix EPS, and this leads to increased retention of both cells and EPS in a growing biofilm. This interaction is predicted to occur between the lectin and the branched side chains present on Psl. Finally, we show that LecB coordinates Psl localization in the biofilm. This constitutes a unique function for LecB and identifies it as a matrix protein that contributes to biofilm structure through EPS interactions.

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix.

Abstract: We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Microneedle Patch-Mediated Treatment of Bacterial Biofilms.

Abstract: Current treatments of bacterial biofilms are limited by the poor penetration of antibiotics through their physical barrier as well as significant off-target toxicity of antibiotics and the induction of antibiotic resistance. Here we report a microneedle patch-mediated treatment for the effective elimination of biofilms by penetrating the biofilm and specifically delivering antibiotics to regions of active growth. We fabricated patches with self-dissolvable microneedles and needle tips loaded with chloramphenicol (CAM)-bearing and gelatinase-sensitive gelatin nanoparticles (CAM@GNPs). During the microneedle patch-mediated treatment, arrays of 225 microneedles simultaneously penetrate the biofilm matrix. Once inside, the microneedles dissolve and uniformly release CAM@GNPs into the surrounding area. In response to the gelatinase produced by the active bacterial community, the CAM@GNPs disassemble and release CAM into these active regions of the biofilm. Moreover, CAM@GNPs exhibited minimal off-target toxicity compared to direct CAM administration, which in turn favors wound healing. Importantly, we found that our microneedle-mediated treatment is more effective in treating Vibrio vulnificus biofilms than drug in free solution. We believe this new treatment strategy can be used to improve the delivery of a wide range of antimicrobial agents to biofilm-contaminated sites.

Confocal Laser Scanning Microscopy for Analysis of Pseudomonas aeruginosa Biofilm Architecture and Matrix Localization.

Abstract: Most microbes can produce surface-associated or suspended aggregates called biofilms, which are encased within a biopolymer-rich matrix. The biofilm matrix provides structural integrity to the aggregates and shields the resident cells against environmental stressors, including antibiotic treatment. Microscopy permits examination of biofilm structure in relation to the spatial localization of important biofilm matrix components. This review highlights microscopic approaches to investigate bacterial biofilm assembly, matrix composition, and localization using Pseudomonas aeruginosa as a model organism. Initial microscopic investigations provided information about the role key matrix components play in elaborating biofilm aggregate structures. Additionally, staining of matrix components using specific labels revealed distinct positioning of matrix components within the aggregates relative to the resident cells. In some cases, it was found that individual matrix components co-localize within aggregates. The methodologies for studying the biofilm matrix are continuing to develop as our studies reveal novel aspects of its composition and function. We additionally describe some outstanding questions and how microscopy might be used to identify the functional aspects of biofilm matrix components.

Conserved Role for Biofilm Matrix Polysaccharides in Candida auris Drug Resistance.

Abstract: Candida auris has emerged as an outbreak pathogen associated with high mortality. Biofilm formation and linked drug resistance are common among Candida species. Drug sequestration by the biofilm matrix accounts for much of the antifungal tolerance. In this study, we examine the biofilm matrix composition and function for a diverse set of C. auris isolates. We show that matrix sequesters nearly 70% of the available triazole antifungal. Like the biofilms formed by other Candida spp., we find that the matrix of C. auris is rich in mannan-glucan polysaccharides and demonstrate that their hydrolysis reduces drug tolerance. This biofilm matrix resistance mechanism appears conserved among Candida species, including C. auris. IMPORTANCECandida auris is an emerging fungal threat linked to poor patient outcomes. The factors responsible for this apparent increase in pathogenicity remain largely unknown. Biofilm formation has been suggested as an important factor for persistence of this organism in patients and the environment. Our findings reveal one mechanism utilized by C. auris to evade the effect of triazole antifungal therapy during biofilm growth. The conservation of the protective biofilm matrix among Candida spp. suggests that is a promising pan-fungal Candida biofilm drug target.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates

Abstract: Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis. Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

A microbiota-generated bile salt induces biofilm formation in Clostridium difficile.

Abstract: Clostridium difficile is a major cause of nosocomial infections. Bacterial persistence in the gut is responsible for infection relapse; sporulation and other unidentified mechanisms contribute to this process. Intestinal bile salts cholate and deoxycholate stimulate spore germination, while deoxycholate kills vegetative cells. Here, we report that sub-lethal concentrations of deoxycholate stimulate biofilm formation, which protects C. difficile from antimicrobial compounds. The biofilm matrix is composed of extracellular DNA and proteinaceous factors that promote biofilm stability. Transcriptomic analysis indicates that deoxycholate induces metabolic pathways and cell envelope reorganization, and represses toxin and spore production. In support of the transcriptomic analysis, we show that global metabolic regulators and an uncharacterized lipoprotein contribute to deoxycholate-induced biofilm formation. Finally, Clostridium scindens enhances biofilm formation of C. difficile by converting cholate into deoxycholate. Together, our results suggest that deoxycholate is an intestinal signal that induces C. difficile persistence and may increase the risk of relapse.

A new tool to attack biofilms: driving magnetic iron-oxide nanoparticles to disrupt the matrix

Abstract: A main feature of biofilms is the self-produced extracellular polymeric substances (EPSs) that act as a protective shield, preventing biocide penetration. We use magnetic iron oxide nanoparticles (MNPs) in combination with magnetic fields to damage the biofilm matrix and cause detachment. A Methicillin-resistant Staphylococcus aureus (MRSA) biofilm strain is used to demonstrate the efficacy of the methodology with different sizes and concentrations of MNPs under AC and DC applied field conditions. We achieve up to a nearly 5 log10 reduction in biofilm bacteria after treatment with 30 mg mL-1 of 11 nm MNPs using a magnetic field. The MNPs cause significant mechanical disruption to the matrix and lead to biofilm dispersal. In addition, using magnetic hyperthermia further affects biofilm damage.

Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence of Streptococcus mutans.

Abstract: Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu_833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors.

Abstract: Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 ± 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (IF) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (IR) remained constant at any pH. The ratio of IF/IR produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.

The Role of Exopolysaccharides in Oral Biofilms.

Abstract: The oral cavity contains a rich consortium of exopolysaccharide-producing microbes. These extracellular polysaccharides comprise a major component of the oral biofilm. Together with extracellular proteins, DNA, and lipids, they form the biofilm matrix, which contributes to bacterial colonization, biofilm formation and maintenance, and pathogenesis. While a number of oral microbes have been studied in detail with regard to biofilm formation and pathogenesis, the exopolysaccharides have been well characterized for only select organisms, namely Streptococcus mutans and Aggregatibacter actinomycetemcomitans. Studies on the exopolysaccharides of other oral organisms, however, are in their infancy. In this review, we present the current research on exopolysaccharides of oral microbes regarding their biosynthesis, regulation, contributions to biofilm formation and stability of the matrix, and immune evasion. In addition, insight into the role of exopolysaccharides in biofilms is highlighted through the evaluation of emerging techniques such as pH probing of biofilm colonies, solid-state nuclear magnetic resonance for macromolecular interactions within biofilms, and super-resolution microscopy analysis of biofilm development. Finally, exopolysaccharide as a potential nutrient source for species within a biofilm is discussed.

Bacterial biofilm destruction by size/surface charge-adaptive micelles.

Abstract: Biofilms formed by pathogenic bacteria are one of the most important reasons for multidrug resistance. One of the major limitations in the biofilm treatment is the existence of intensive matrices, which greatly block the diffusion of antimicrobial agents. In the current study, we designed poly(aspartamide)-derived micelles self-assembled from cationic copolymers with azithromycin-conjugated and pH-sensitive copolymers, followed by loading cis-aconityl-d-tyrosine (CA-Tyr) via electrostatic interactions. In response to the acidic microenvironment of the biofilm matrix, the hydrophilic transition of the pH-sensitive copolymers and the removal of CA-Tyr led to a sharp decrease in micelle size from 107 nm to 54 nm and a rapid shift in their zeta potential from -11.7 mV to +26.4 mV, which facilitated the penetration of the micelles into biofilms. The acid-labile release of d-tyrosine disintegrated the biofilm matrix, and the lipase-triggered release of azithromycin eradicated the bacteria in the biofilms. An in vitro test was performed on pre-established P. aeruginosa biofilms in microwells, while biofilms grown on catheters were surgically implanted in rats for in vivo evaluation. The results demonstrated the capabilities of the size/surface charge-adaptive micelles in the intensive infiltration in the biofilm matrix and spatiotemporal release of biofilm dispersion and antibacterial agents for the comprehensive treatment of biofilm-relevant infections.

Iron Homeostasis in Bacillus subtilis Requires Siderophore Production and Biofilm Formation.

Abstract: Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in Bacillus subtilis We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in B. subtilis, both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria.IMPORTANCE Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.

Targeting Bacterial Biofilms by the Green Tea Polyphenol EGCG.

Abstract: Bacterial biofilms are multicellular aggregates in which cells are embedded in an extracellular matrix of self-produced biopolymers. Being refractory to antibiotic treatment and host immune systems, biofilms are involved in most chronic infections, and anti-biofilm agents are being searched for urgently. Epigallocatechin-3-gallate (EGCG) was recently shown to act against biofilms by strongly interfering with the assembly of amyloid fibres and the production of phosphoethanolamin-modified cellulose fibrils. Mechanistically, this includes a direct inhibition of the fibre assembly, but also triggers a cell envelope stress response that down-regulates the synthesis of these widely occurring biofilm matrix polymers. Based on its anti-amyloidogenic properties, EGCG seems useful against biofilms involved in cariogenesis or chronic wound infection. However, EGCG seems inefficient against or may even sometimes promote biofilms which rely on other types of matrix polymers, suggesting that searching for ‘magic bullet’ anti-biofilm agents is an unrealistic goal. Combining molecular and ecophysiological aspects in this review also illustrates why plants control the formation of biofilms on their surfaces by producing anti-amyloidogenic compounds such as EGCG. These agents are not only helpful in combating certain biofilms in chronic infections but even seem effective against the toxic amyloids associated with neuropathological diseases.

Treatment with the Pseudomonas aeruginosa Glycoside Hydrolase PslG Combats Wound Infection by Improving Antibiotic Efficacy and Host Innate Immune Activity.

Abstract: Pseudomonas aeruginosa is an opportunistic, nosocomial bacterial pathogen that forms persistent infections due to the formation of protective communities, known as biofilms. Once the biofilm is formed, the bacteria embedded within it are recalcitrant to antimicrobial treatment and host immune defenses. Moreover, the presence of biofilms in wounds is correlated with chronic infection and delayed healing. The current standard of care for chronic wound infections typically involves physical disruption of the biofilm via debridement and subsequent antimicrobial treatment. The glycoside hydrolases PelAh and PslGh have been demonstrated in vitro to disrupt biofilm integrity through degradation of the key biofilm matrix exopolysaccharides Pel and Psl, respectively. Herein, we demonstrate that PslGh hydrolase therapy is a promising strategy for controlling P. aeruginosa wound infections. Hydrolase treatment of P. aeruginosa biofilms resulted in increased antibiotic efficacy and penetration into the biofilm. PslGh treatment of P. aeruginosa biofilms also improved innate immune activity leading to greater complement deposition, neutrophil phagocytosis, and neutrophil reactive oxygen species production. Furthermore, when P. aeruginosa-infected wounds were treated with a combination of PslGh and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslGh alone. This study demonstrates that PelAh and PslGh have promising therapeutic potential and that PslGh may aid in the treatment of P. aeruginosa wound infections.

5-Dodecanolide interferes with biofilm formation and reduces the virulence of Methicillin-resistant Staphylococcus aureus (MRSA) through up regulation of agr system.

Abstract: Methicillin resistant Staphylococcus aureus (MRSA) is a predominant human pathogen with high morbidity that is listed in the WHO high priority pathogen list. Being a primary cause of persistent human infections, biofilm forming ability of S. aureus plays a pivotal role in the development of antibiotic resistance. Hence, targeting biofilm is an alternative strategy to fight bacterial infections. The present study for the first time demonstrates the non-antibacterial biofilm inhibitory efficacy of 5-Dodecanolide (DD) against ATCC strain and clinical isolates of S. aureus. In addition, DD is able to inhibit adherence of MRSA on human plasma coated Titanium surface. Further, treatment with DD significantly reduced the eDNA synthesis, autoaggregation, staphyloxanthin biosynthesis and ring biofilm formation. Reduction in staphyloxanthin in turn increased the susceptibility of MRSA to healthy human blood and H2O2 exposure. Quantitative PCR analysis revealed the induced expression of agrA and agrC upon DD treatment. This resulted down regulation of genes involved in biofilm formation such as fnbA and fnbB and up regulation of RNAIII, hld, psmα and genes involved in biofilm matrix degradation such as aur and nuc. Inefficacy of DD on the biofilm formation of agr mutant further validated the agr mediated antibiofilm potential of DD. Notably, DD was efficient in reducing the in vivo colonization of MRSA in Caenorhabditis elegans. Results of gene expression studies and physiological assays unveiled the agr mediated antibiofilm efficacy of DD.

Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.

Abstract: The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm.

Small Is Mighty-Chemical Communication Systems in Pseudomonas aeruginosa.

Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of acute and chronic infections. Usually a commensal on the host body, P. aeruginosa is capable of transforming into a virulent pathogen upon sensing favorable changes in the host immune system or stress cues. P. aeruginosa infections are hard to eradicate, because this pathogen has developed strong resistance to most conventional antibiotics; in addition, in chronic infections it commonly forms a biofilm matrix, which provides bacterial cells a protected environment to withstand various stresses including antibiotics. Given its importance as a human pathogen and its notorious antimicrobial tolerance, P. aeruginosa has been the subject of intensive investigations internationally. Research progress over the last two decades has unveiled a range of chemical communication systems in this pathogen. These diversified chemical communication systems endow P. aeruginosa a superb ability and remarkable flexibility to coordinate and modulate accordingly the transcriptional expression of various sets of genes associated with virulence and other physiologic activities in response to environmental changes. A fair understanding of the chemical signaling mechanisms with which P. aeruginosa governs virulence gene expression may hold the key to developing alternative therapeutic interventions that control and prevent bacterial infections.

Effects of a Novel Probiotic Combination on Pathogenic Bacterial-Fungal Polymicrobial Biofilms

Abstract: Dysbiosis of the gut microbiome has been implicated in inflammatory bowel diseases. We have shown that levels of Candida tropicalis, along with those of Escherichia coli and Serratia marcescens, are significantly elevated in Crohn's disease (CD) patients. Here, we evaluated the ability of a novel probiotic to prevent and treat polymicrobial biofilms (PMB) formed by C. tropicalis with E. coli and S. marcescens Since Candida albicans has been reported to be elevated in CD patients, we investigated the interactions of C. albicans with these bacterial species in biofilm formation. We determined whether the interaction between Candida spp. and bacteria is specific by using Trichosporon inkin and Saccharomyces fibuligera as comparators. Additionally, the effects of probiotics on C. albicans germination and biofilm formation were determined. To determine the ability of the probiotic to prevent or treat mature biofilms, probiotic filtrate was added to the PMB at early (prevention) and mature (treatment) phases. Biofilm thickness and architecture were assessed by confocal scanning laser microscopy. The effects of the probiotic on germination were evaluated in the presence of serum. Exposure of C. tropicalis PMB to probiotic filtrate reduced biofilm matrix, decreased thickness, and inhibited hyphal formation. We showed that C. albicans or C. tropicalis formed significantly thicker PMB than control biofilms, indicating that this interaction is Candida specific. Treatment with probiotic filtrate inhibited C. albicans germination and prevented/treated C. albicans PMB. The designed probiotic may have utility in the management of biofilm-associated gastrointestinal diseases such as Crohn's and colorectal cancer.IMPORTANCE The effects of diversity of the gut microbiome on inflammation have centered mainly on bacterial flora. Recent research has implicated fungal species and their interactions with other organisms in the inflammatory process. New ways to restore microbial balance in the gut are being explored. Our goal was to identify beneficial probiotic strains that would antagonize these fungal and bacterial pathogens that are elevated in the inflamed gut, and which also have antibiofilm activity. Fungus-bacterium correlation analysis allowed us to identify candidate probiotic species that can antagonize microbial pathogens, which we subsequently incorporated into a novel probiotic formulation. Amylase, which is known to have some antibiofilm activity, was also added to the probiotic mixture. This novel probiotic may have utility for the management of inflammatory bowel diseases by disrupting polymicrobial biofilm formation.

Heterogeneity in respiratory electron transfer and adaptive iron utilization in a bacterial biofilm.

Abstract: In Bacillus subtilis, robust biofilm formation requires large quantities of ferric iron. Here we show that this process requires preferential production of a siderophore precursor, 2,3-dihydroxybenzoate, instead of the siderophore bacillibactin. A large proportion of iron is associated extracellularly with the biofilm matrix. The biofilms are conductive, with extracellular iron potentially acting as electron acceptor. A relatively small proportion of ferric iron is internalized and boosts production of iron-containing enzymes involved in respiratory electron transfer and establishing strong membrane potential, which is key to biofilm matrix production. Our study highlights metabolic diversity and versatile energy generation strategies within B. subtilis biofilms.